16-alpha oxy-delta1, 4-pregnadienes



2,806,043 IG-ALPHA OXY-A -PREGNADIENES Seymour Bernstein and- William S. Allen, Pearl River, N. Y., assignors to American Cyanamid Company, New York, N. Y., a corporation of Maine N Drawing. Application June 11, 1956,

a Serial No. 590,791

4 Claims. (Cl. 260-39145) This invention relates to new A -steroids. More particularly, it relates to steroids containing the A -pregnadiene-3,l1,20-trione moiety.

, Recently a number of steroids of the pregnene and pregnadiene series, such as hydrocortisone and l-dehydrohydrocortisone, have become important therapeutic CHZOB.

MU... B

in which R and R are hydrogen or a lower alkanoyl radical.

The compounds of the present invention are crystalline solids, having a definite melting point. They are, in general, soluble in the usual organic solvents.

The present compounds are prepared from the corresponding 16a,21-di-loweralkanoyl derivatives of 11 6,1701- dihydroxy-l,4-pregnadiene-3,20-dione by oxidation with chromium trioxide. Following oxidation, the lower alkanoyl groups can be removed by saponification.

The starting material used in the process of the present invention can be prepared either chemically or biologically. The example hereinafter describes the preparation of 16a,21-di-loweralkanoyl derivatives of 115,17a-dihydroxyl,4-pregnadiene-3,20-dione by a fermentative method using, for example, the fungus Corynebacterium simplex. The substrate or starting material used with the fungus is 115,16u,17m,21-tetrahydroxy-4-pregnene-3,ZO-dione.

In carrying out the process of the present invention a 16a,2l-di-1oweralkanoyl ester'of 11fl,17a-dihydroxy-l,4- pregnadiene-3,20-dione is dissolved in a solvent such as pyridine. A solution of an oxidizing agent such as chromium trioxide in pyridine is added, and the mixture is allowed to stand at room temperature. After the reaction is complete, a further solvent is added, such as a lower alkyl alcohol, and the solvents are removed by evaporation under reduced pressure. The product is purified by methods well known to those skilled in the art.

The following example illustrates the process of the present invention:

2,806,043 Patented Sept. 10, 1957 Example A Trypticase soy agar test tube slant was-washed with 5 ml. of sterile water, and the resulting cell suspension of Corynebacterium simplex was used to inoculate 100 ml. of sterile Trypticase soy broth medium in a 500 ml. Erlenmeyer flask. This mixture was incubated with shaking at 37 C. for eight hours. Twenty-five 500 ml. Erlenmeyer flasks, each containing 100 ml. of sterile Trypticase soy broth medium without glycerol, were each inoculated with 1 ml. of the eight-hour-old inoculum. These flasks were incubated at 32 C. for 40 hours. At this time, 40 mg. of l15,1611,1fZa,21-tetrahydroxy-4- 'pregnene-3,20-dione dissolved in 4ml. of ethanol were added to each flask, and the fermentation was continued for eight hours at 32 C. The contents of all 25 flasks were pooled, giving a mash of pH 8.1.

The pooled beer, after harvest, was extracted once with 3 liters of methylene chloride and three times with 2-liter portions of methylene chloride. The combined extract was washed once with saturated saline and evaporated to dryness under reduced pressure. This gave 509 mg. of oily residue, which was dissolved in 1.5 ml. of the stationary phase from the system, 3 parts ethyl acetate:2 parts petroleum ether (100 C.):3 parts methanol:2 parts water, and mixed with 3 g. of diatomaceous earth. This impregnated diatomaceous earth was then packed on top of a 15x35 cm. glass column containing 25 g. of diatomaceous earth impregnated with 12.5 ml. of the stationary phase from the above system. The 11 3,160,17a,21-tetrahydroxy- 1,4-pregnadiene-3,ZO-dione was then eluted with the mobile phase from the above system, crude solid. This was crystallized from acetone-petroleum ether (6070 C.) to give 56 mg. Recrystallization from the same solvent gave a melting point to 202-205 C. (block); melting point, 229-231 C. (capillary method).

The physical and chemical properties of the compound were those for 115,16a,17a,21-tetrahydroxy-1,4-pregnadiene-3,20-dione.

A mixture of 2.5 g. 1119,16a,17a,21-tetrahydroxy-l,4- pregnadiene-3,20-dione, obtained above, in ml. of pyridine containing 25 ml. of acetic anhydride was allowed to stand overnight at room temperature, after which the solution was evaporated to dryness under reduced pressure. The solid residue was crystallized from ethyl acetate-petroleum ether (90-100 C.), giving 16u,21-diacetoxy-11B,17a dihydroxy 1,4 pregnadiene- 3,20-dione in two fractions: (a) melting point 215-217 C., Weight 1.03 g. and (b) melting point 217219 C.,

weight 1.2 g. Recrystallization of a portion of (b) from the same solvent pair did not alter the melting point.

Analysis.-Calcd for CzsHaaOa (460.51): C, 65.20; H, 7.00. Found: C, 64.88; H, 7.31.

A solution of 200 mg. of l6m,21-diacetoxy-115,170:- dihydroxy-1,4-pregnadiene-3,20-dione in 10 ml. of dry pyridine was treated with a solution of mg. of chromium trioxide in 8 ml. of pyridine and the mixture allowed to stand overnight at room temperature. Then 10 ml. of methanol was added and the solvents were evaporated oil under reduced pressure. Water was added to the dry residue, and the mixture was extracted with five-200 ml. portions of ethyl acetate. The com-' bined extract was dried over anhydrous sodium sulfate and evaporated to a solid residue of l6a,21-diacetoxy 17a-hydroxy-1,4-pregnadiene-3,l1,20-trione, which on crystallization from ethyl acetate-petroleum ether (90100. C.) gave 128 mg., melting point 216-217 C. (64%). Repeated crystallization from the same solgiving 207 mg. of

3 vent pair changed the melting point to 208-209 C., [M -{433 C. (methanol).

Analysis-Calcd for CzsHsoOs (458.49): C, 65.49; H, 6.60. Found: C, 65.21;.H, 6.68.

We claim: 7 5 1. Compounds having the general formula 10 0 I-OR in which R and R are members of the group consisting of hydrogen and lower alkanoyl radicals. l

2. Compounds in accordance with claim 1 inrwhich R- and R are lower alkanoyl radicals.

3. The compound 16a,21-d1'acetoxy-17a-hydroxy-L4- pregnadiene-3,11-20-trione.

4. The compound 16a,1741,2Ltrihydroxy-lA-pregnadiene-3,11,20-trione.

References Cited in the file of this patent V UNITED STATES PATENTS 2,736,734 Sarett Feb. 28, 1956 2,767,199 Djerassi Oct. 16, 1956 2,773,058 Bernstein Dec. 4, 1956 2,773,080 Bernstein Dec. 4, 1956 2,774,775 Korman Dec. 18, 1956 

